Epitranscriptomics explores levels and evolution of RNA modifications as a response from intra and inter- cellular stimuli. These modifications and the levels at which they are present are emerging as important regulators of biological processes. Perturbations such as chemical toxicants exposure can alter the patterns of specific modifications on RNA sub-types, and lead to new non-natural modifications. Moreover, given their variety, specificity and their relationship to function and structure stability, RNA modifications are potentially valuable biomarkers of disease and treatment progression, especially given that modified nucleosides are relatively stable even after RNA degradation, and can be released from cells into bodily fluids. However the exploration of RNA and in particular RNA modifications for medical use requires the development of novel high-throughput methods and technologies. The long term objective of this study is to facilitate epitranscriptomics research into RNA modifications and their impact on health and disease. The immediate hurdle, and the goal of this application, is to develop extremely sensitive, high-throughput analytical methods to screening large variety chemical toxicants in the context of common and novel RNA modifications during human stem cell differentiation, which can be readily disseminated to the RNA research community. The test case biological system is based on human induced pluripotent stem cell derived nervous system cell differentiation that will be exposed to two different chemical toxicants, nicotine and bisphenol A (BPA). Aim 1 will use RNA modified nucleoside standards to develop highly sensitive and robust method of analysis. This will allow the generation of epitranscriptome baseline during differentiation of human pluripotent stem cells into frontal cerebral cortex neurons and glial cells. The final development will result from the combination of two technologies, UHPLC and MS that will be optimized for high resolution separation and highly sensitive detection of regulatory RNA modifications. The tools developed will be tested on differentiated anterior cerebral cortex neurons cells and RNA modifications will be characterized and abundances quantified during the different stages of differentiation. In Aim 2, tools developed during Aim 1 will be adapted to assess changes in the epitranscriptome that may be playing a significant role on differentiated forebrain cells, as a result exposure to chemical toxicants. During this aim we will specifically study forebrain cells that will be exposed to different nicotine and BPA concentrations and compared to vehicle controls. The results of the methods development will be a high-throughput, extremely sensitive and accurate technology for the analysis of a wide range of chemistries and abundances of natural and non-natural RNA modifications of the epitranscriptome, applied to human neural stem cell differentiation. The technology will allow for rapid temporal assessment of changes in the epitranscriptome.